Protease inhibitors and method for preparation

ABSTRACT

1. A PROCESS FOR PREPARING A PREPARATION CONTAINING PROTEASE INHIBITORS WHICH COMPRISES CULTIVATING STREPTOMYCES VIOLASCENS, NRRL 3859, UNDER AEROBIC CONDITIONS IN A NUTRIENT MEDIUM CONTAINING SOURCES OF CARBON, NITROGEN, AND NUTRIENT INORGANC SALTS IN ASSIMILABLE FORM, FOR A PERIOD OF ABOUT 10 TO 100 HOURS UNTIL A SUFFICIENT AMOUNT OF PROTEASE INHIBITORS IS PRODUCED, SAID MEDIUM HAVING A PH, PRIOR TO INOCULATION, RANGING BETWEEN ABOUT 5 AND 8, AND A TEMPERATURE DURING TERMENTATION RANGING FROM ABOUT 10* TO ABOUT 40*C.

United States Patent (Mike 3,849,552 PROTEASE INHIBITORS AND METHOD FORPREPARATION Arne Gosta Jonsson, Sodertalje, and Nils Torsten LennartTorstensson, Uppsala, Sweden, assignors to Astra Lakemedel Aktiebolag,Sodertalje, Sweden N Drawing. Continuation of abandoned application Ser.No. 128,493, Mar. 26, 1971. This application June 21, 1973, Ser. No.372,208 Claims priority, application Great Britain, Apr. 1, 1970,15,530/70; Jan. 28, 1971, 3,426/71 Int. Cl. A61k 21/00 US. Cl. 424-115 2Claims ABSTRACT OF THE DISCLOSURE A new strain of microorganismbelonging to the order Actinomycetales, genus Streptomyces anddesignated as Streptomyces violascens is capable of producing proteaseinhibitors having unique characteristics and a wide variety ofbiological and medical uses. These enzyme inhibitors are prepared bycultivating the newly found microorganism under aerobic submergedconditions.

This is a continuation of application Ser. No. 128,493, filed Mar. 26,1971, now abandoned.

The present invention relates to enzyme inhibitors, and moreparticularly, to protease inhibitors produced by cultivating a newlydiscovered strain of the genus Streptomyces, or mutants thereof. Thisinvention also relates tomethods for the preparation, isolation andpurification of these novel protease inhibitors.

The increasing use of enzymes and enzyme preparations in various fieldsof technology has caused a corresponding need for agents capable ofcontrolling the activity of enzymatic processes. Previously, enzymeinhibitors, and in particular inhibitors of proteolytic enzymes, havegenerally been prepared from mammalian tissue. US. Pat. No. 3,532,724discloses an inhibitor of the protease renin which inhibitor is preparedfrom mammalian kidney tissue. The preparation of these inhibitors frommammalian tissue has the obvious drawback of limiting the amount ofmaterial which can be produced to such an extent that commercialproduction is not feasible. Accordingly, while there is a great need forenzyme controlling agents, particularly protease inhibitors,

production of such agents by fermentation on a large scale has notheretofore been realized.

Protease inhibitors are useful in various fields of medicine. Proteaseinhibitors are eifective in the treatment of various states of shock; inthe treatment of pancreatitis; in the treatment of blood clots; and inorder to prevent the formation of blood clots. Protease inhibitors arealso useful in obstetrics and heart surgery. It is known that thespontaneously occurring disintegration of organs and tissues, such asliver, kidney, heart, pancreas, lung, skin and bonemarrow, after deathmay be suppressed by substances capable of inhibiting proteolyticactivity which are obtained from mammalian tissue or plants. As aresult, protease inhibitors are useful to preserve organs and tissuesduring their transplantation. Similarly, certain nutrition agents may bepreserved from disintegration and destruction caused by proteolyticenzymes by treating them with protease inhibitors.

3,849,552 Patented Nov. 19, 1974 A field of particular interest wherethe protease inhibitors according to the present invention may be usedis the treatment of states of elevated fibrinolytic activity in bodyfluids, such as blood and urine in mammals including man. Elevatedfibrinolytic activity may result from pathologic conditions, such aspancreatitis, but may also be externally induced, for example byadministration of fibrinolytic agents such as Protease I described byBergkvist in US. Pat. No. 3,281,331. Protease I may be used clinicallyfor dissolution of blood clots and must be administered in carefullycalculated dosages. Heretofore, it has not been possible to counteractthe proteolytic activity of Protease I once it has been administered tothe patient in excessive amounts. The protease inhibitor according tothe present invention has surprisingly appeared to be active alsoagainst Protease I and may be used as an antidote in cases of overdosagewith Protease I.

Protease inhibitors are also useful in other fields, for example inenzymology, where they serve as natural tools with which to probe theactive centers of proteases, for isolation of enzymes, and forpurification of enzymes in a simple one-step procedure. Inhibitors mayalso be of importance in ecological studies. In technical processes,where a rapid and mild inhibition of proteolytic enzymes is wanted,there is a need for cheap inhibitors. Another field where proteaseinhibition may be used is food technology, particularly for fishprocessing, for example in the production of fishmeal and productsderived therefrom.

The present invention makes possible large scale production of proteaseinhibitors with valuable properties. It has been found that a newlydiscovered strain of the genus Streptomyces, and mutants thereof, may becultimated to produce enzyme controlling agents, in particular proteaseinhibitors.

The microorganism used in the production of the protease inhibitors ofthe present invention has been isolated from a soil sample collected inArkelstorp, Sweden. Taxonomic observations indicate that thismicroorganism belongs to the order Actinomycetales, genus Streptomyces.It was identified as Streptomyces violascens. A subculture of thisvariety can be obtained from the permanent collec'tion of the UnitedStates Department of Agriculture, Agricultural Research Service,Northern Utilization Research and Development Division, 1815 NorthPeoria Street, Peoria, Ill. 61604, USA. The accession number for thesubsulture in this repository is NRRL 3859.

The present invention relates to a strain Streptomyces violascens (NRRL3859) and mutants thereof, heretofore unknown, which are capable ofproducing protease inhibitors showing the unique characteristics andqualities hereinafter described. The invention also relates to a methodof producing preparation containing protease inhibitors obtained bycultivating the new strain Streptomyces violascens (NRRL 3859) underaerobic conditions in a suitable nutrient medium. The strain may also becultivated under surface conditions.

In a preferred embodiment of the process which suitably may be used forthe production of the protease inhibitors of the present invention, themicroorganism is grown under aerobic submerged conditions in a nutrientmedium which comprises an assimilable source of carbon, nitrogen andinorganic salts. Cultivation conditions such as time, temperature andhydrogen ion concentration may be varied broadly without substantiallyeffecting the growth of the microorganism. At the conclusion of thegrowth period, the protease inhibitor material may be re covered fromthe mash by a number of methods, such as filtering, centrifugation ofthe mash, precipitation of impurities with acetone, and freeze-drying orspray-drying the resulting solution. The protease inhibitors may befurther recovered from the preparation by dialyzation, ultrafiltrationand gel filtrations.

A wide variety of substances may be used as sources of carbon in thenutrient medium. These carbon sources may be either soluble or insolublein 'water, it being desirable only that the compounds used be readilyassimilable by the organism. Examples of suitable carbon sources includemonosaccharides such as glucose, fructose, and pentoses such asarabinose, disaccharides such as sucrose, maltose and lactose,trisaccharides such as raffinoe, and polysaccharides such as starch andcellulose. Higher alcohols such as glycerol and inositol are alsosuitable carbon sources.

Nitrogen in an assimilable form may be provided by animal or vegetableproteins such as soybean meal, casein, peptones, polypeptides, aminoacids, or the like.

The nutrient inorganic salts which can be incorporated into the mediummay be selected from the group consisting of salts capable of yieldingions such as ammonium, sodium, potassium, calcium, magnesium, phosphate,chloride, sulphate, nitrate and the like. For optimum growth of themicroorganism, it is preferred to add a combination of K HPO NaNO andMgSO -7 H O to the culture medium. Essential trace elements may also beincluded in the culture medium. Trace elements are, however, usuallypresent as impurities in other components in the culture medium.

For maximum growth and production of the new microorganism of thepresent invention the culture medium, prior to inoculation, should beadjusted to a pH between about and 8. The pH at the end of the growthperiod will depend on the buffering substances present and on theinitial pH. Adjustment of the pH may be accomplished with any suitableacid such as hydrochloric acid or with phosphate, citrate or acetatebuffer or any other suitable material which would bring the pH withinthedesired range.

The temperature of the nutrient medium during fermentation of theactinomycete may be maintained within the interval from about 10 C. toabout 40 C. and may advantageously be maintained at 30 C. or lower,preferably from about C. to about 30 C.

The time required for the production of a maximum PROTEASE SCREENINGPROCEDURE? The following method was used for screening for'proteaseinhibitors. A double layer of agar inPetri dishes (90 mm.) was used. Thebottom layer consisted of skim-milk agar, which contained skim-milkpowder (Semper AB, Stockholm, Sweden), 1 and agar No. 3'(Oxoid), 12 g./liter of 0.02 M phosphate buffer, pH 7.5. After spreading of 0.1 ml. ofdiluted sample on the surface, the plate was incubated for 2 days at 24C. After this time, it was cooled at 2 C. for 1 hr. Protease fromAlternaria tenuissima, 1 enzyme unit/liter (Jtinsson, Appl. Microbiol.15 (1967) 319-324) was sterilized by filtration and added to an agarsolution (43 C.), containing 12 g. of agar per liter of the samephosphate buffer, to give a protease activity of 0.1 enzyme unit/ liter.Thereafter, this agar was poured on top of the bottom layer. The platewas incubated for additional 24 hrs. at 24 C. The skim milk agar thenturned transparent, except around colonies producing proteaseinhibitors. These colonies were used for inoculation of 200 ml.Erlenmeyer flasks, containing 25 ml. of Nutrient Broth (Oxoid). Theflasks were incubated at 24 C. as shake cultures (180 rev./min., model G10 shakes; New Brunswick Scientific Co., New Brunswick, N.J.). After 3days, the trypsin inhibition activity in the culture filtrate wasestimated as follows:

Crystallized salt free trypsin (3215 NE. Units per mg., ArmourPharmaceutical Company Ltd., Eastbourne, England) was used for theestimation of inhibitor effect. 0.2 ml. culture filtrate from the shakeculture and 0.8 ml. trypsin solution, containing about 5 g. trypsin, wasincubated for min. at 24 C. Blanks were incubated with 0.8 ml. trypsinsolution and 0.2 ml. distilled water. The remaining free trypsin, notinhibited, was estimated as caseolytic activity at pH 9.5 by amodification of Ansons procedure (Itinsson, Appl. Microbial. 15 (1967)319- 324). Because the correlation between inhibition and concentrationof inhibitor is linear only to a certain limit, dilutions of culturefiltrate to give about percent inhibition of the trypsin were used.Inhibitor activity was calculated as the amount of trypsin g.) that wasinhibited to 50 percent by 1 ,ul. of culture filtrate. The data providedin this disclosure relating to inhibitor activity refer to this 50percent inhibition.

A similar method was used for estimation of inhibition of otherproteases, for example, chymotrypsin (crystalline alpha-chymotrypsinfrom bovine pancreas, BDH).

TAXONOMIC IDENTIFICATION OF THE MICROORGANISM The recommendationsaccording to Waksman, The Actinomycetes (1950) and the system ofclassification in Bergeys Manual of Determinative Bacteriology, VIII Ed.(1957) were followed for the taxonomic identification of themicroorganism. For identification of species, the recommendations of TheInternational Streptomyces Project (Shirling & Gottlieb, Int. J. Syst.Bacteriol. 16 (1966) 313-340; Int. J. Syst. Bacteriol, 18 (1968) 69-189;Int. J. Syst. Bacteriol. 18 (1968) 279-399; Int. J. Syst. Bacteriol. 19(1969) 391-512); were followed. The following results were obtained:

(1) 'The microorganism grew with a branching mycelium-like structure andthe hyphae were about 1.0 ,um. in diameter. The vegetative myceliumremained undivided. Conidia were produced in chains from aerial hyphae.

(2) The organism had the following properties at cultivation on variousmedia:

(a) Spore morphology: Mature spore chains comprised 10 to 50 or morespores per chain. This morphology was seen on yeast-malt, oatmeal,salt-starch, and glycerolasparagine agar. Spore surface was spiny.

(b) Color of colony: Aerial mass color in the white color-series onyeast-malt agar, salts-starch agar, and glycerol-asparagine agar and inthe violet color series on o'atmeal'agar.

(c) Reverse side of colony: No distinct pigments (pale yellow to lightbrown) on yeast-malt agar, oatmeal agar, salts-starch agar, andglycerol-asparagine agar.

.(d) Color in medium: Melanoid pigments were found in peptone-yeast ironagar, tyrosine agar, and tryptoneyeast broth. Pigments other thanmelanoids were not produced in yeast-malt agar, oatmeal agar,salts-starch agar, and glycerol-asparagine agar.

(e) Carbon utilization: D-glucose, L-arabinose, i-inositol, D-fructose,raflinose, and cellulose were utilized for growth. No growth or onlytraces of growth on sucrose, D-xylose, D-mannitol or rhamnose.

(f) Appearance on further media is given in Table I.

TABLE I Appearance of the strain of Streptomyces on various media MediumAppearance of colonies Czapeks agar Separate white colonies.Glucose-asparagine agar Separate white colonies. Starch-nitrate agarSeparate grayish-beige colonies. Nutrient agar Separate grayish-beigecolonies. Sabouraud agar Separate grayish-beige colonies. PotatoeSeparate live-green colonies. Carrot Separate live-green colonies.

3) Gelatine is not liquefied. Gram stain: positive. Not acid-fast.

(4) The growth velocity of mycelium was maximal at about 28 C. At 5 C.there is a slight growth whereas at 40 C. there is no growth ofmycelium.

(5) The maximal production of protease inhibitor occurred at a lowertemperature than the maximal growth of mycelium.

(6) The trypsin inhibiting capacity of the isolated microorganism of thepresent invention, measured as g. trypsin inhibited to 50% by 1 l.culture filtrate, was 0.1 pg./,u.l.

These taxonomic observations lead to the determination that the newmicroorganism belongs to the order Actinomiycetales, genus Streptomyces.It is identified as Streptomyces violascens.

Various types of media were tested in studies of the inhibitorproduction from the Streptomyces strain of the present invention.Samples were withdrawn and assayed after 2, 4 and 8 days of incubation.Three synthetic media, used for actinomycetes, gave little inhibitoractivity, 0.04 g. of trypsin inhibited to 50% per l., or less, after 4days of incubation: (1) Czapeks sucrose medium; (2) starch-nitratemedium; and (3) synthetic lactate medium (Waksman, 1950, pp. 193-197,No. 18, 19', 26). Growth was also relatively sparse in these media withthe exception of the starch-nitrate medium. Higher yields were attainedin nutrient broth (Oxoid) glucose-peptone medium (Sabouraud), and in amilk medium (Dion, 1950), a maximum of about 0.3 g. of trypsin inhibitedto 50% per l. after 4 days.

The highest yields were attained from a medium containing spray-driedprotein powder (Bast Bovine Cell Tissue, C. E. Basts Successors Ltd.,Copenhagen, Denmark) which contained (g. per liter in distilled water):protein powder, 18; glucose, K HPO 1.0; KCl, 0.5; NaNo 3.0; MgSO.;. 7HO, 0.5; and FeSO .7H O, 0.01. The etfect of various concentrations ofprotein powder in the me dium was, therefore, studied. Thisconcentration of protein powder resulted in an inhibitor concentrationof 1.2 g. of trypsin inhibited to 50% per l. of culture filtrate after72 hrs. of incubation. The other concentrations studied gave much lowerinhibitor production. The typical shift in pH in connection with growthin this medium is seen in Table II. Similar yields were obtained whenKCl, MgSO and FeSO were excluded.

TAB LE II [Production of inhibitor in a protein powder medium]Incubation time The production of protease inhibitor was also performedin 8-liter fermentors. The resulting inhibitor activity was found to belower in these fermentors than in shake-cultures with the same medium.This may, at least partly, be caused by the rapid increase in pHobserved in fermentors. When pH was prevented from increasing over pH 5by automatic additions of 0.5 -M HCl, higher activity was observed.After a lag phase of about 8 hrs. the actinomycete grew for additional28 hrs. The inhibitor production almost parallelled growth and reached amaximum concentration of 0.9 pg. (trypsin per pl. after 29 hrs. ofincubation. The automatic foaming control required approximately 300 ml.of antifoam emulsion when pH was allowed to vary freely, but at pH 5relatively small amounts were used of about ml. per fermentor after 40hrs.

The components of the protein powder medium described above were usedfor cultivation at various pH values, but dissolved in acitrate-phosphate butter (0.02 and 0.04 M in regard to citric acid andNa HPO respectively). PH was adjusted to 2.8-9.9 with 6 N HCl or 4 NNaOH.

The effect of starting pH in the medium on growth and production ofprotease inhibitor as estimated after hrs. of incubation is seen inTable III. As is seen in Table III a starting pH between 6 and 7 wasmost favorable for the process. However, final pH was 1 to 2 unitshigher at the end of the experiment, despite the buffering capacity ofthe medium. PH control to pH 5 during the latter part of the processseemed favorable in fermentors, where maximum activity was attainedafter 28 hrs. of cultivation.

TAB LE III [Etfeet of starting pH on growth and production on inhibitorin a protein powder medium] The eifect of temperature on production ofthe protease inhibitor in quiescent flasks is shown in Table IV.

TAB LE IV [Effect of temperature on production of inhibitor in a proteinpowder medium after 10 and 24 days of surface growth] Trypsin inhibition(r -h 10 days Temperature Nor As seen in Table IV the maximum inhibitoractivity was attained at about 15 C. to 20 C. This was particularly soat the earlier harvest, after 10 days of surface growth. At 37 C.,growth and production were close to zero.

The purification procedure for the protease inhibitors may be carriedout by removing of the mycelium from the culture by suction filtering,freeze-drying of the culture filtrate, and methanol extraction of thefreeze-dried powder. After an addition of distilled water to the extractthe methanol was evaporated by vacuum, and the water solution wasdialyzed and ultrafiltered. Finally the fraction in the ultrafiltratewhich contains the inhibitors was separated by gel filtration into twofractions, one fraction containing a trypsin inhibitor withoutchymotrypsin inhibiting capacity and one fraction containing achymotrypsin inhibitor without trypsin inhibiting capacity. Both theprotease inhibitors of the present invention were found to be dialyzableand to be able to inhibit the activity of Protease I from Aspergillusoryzae (Bergkvist, US. Pat. No. 3,281,331). They inhibited partly theproteolytic activity in fish and fish products. No bacteriostatic orfungistatic etfects were observed. The chymotrypsin inhibitor inhibitedoc-, [i-, 'y and e-chymotrypsin. Both inhibitors were stable at heatingto 100 C. for 20 min. They were also stable in solutions between pH 2and pH 9 and the trypsin inhibitor showed no isoelectric point under pH10. The molecular weights were about 1,400, and 1,000 or lower, for thetrypsin and chymotrypsin inhibitor, respectively, as estimated by gelfiltration on Sephadcx G-50.

In clinical practice, a pharmaceutical preparation containing atherapeutically effective dose of the protease inhibitors according tothe present invention may be administered orally or parenterally.Dosages must be carefully adjusted depending on the individualrequirements of each patient.

The following Examples are provided only to illustrate the invention andare not intended to limit its scope in any way.

CULTIVATION OF THE MICROORGANISM NRRL 3859 Example 1 A suspension of themicroorganism NRRL 3859 and nutrient broth (Oxoid Ltd., London, England)was inoculated on a 10-ml. nutrient agar slant (Oxoid). After days at 28C., to mm. portions of the culture were transferred to 200 ml.Erlenmeyer flasks with 50 ml. of nutrient broth (Oxoid) and incubated at24 C. on a rotary shaker (180 rev./min.). After 4 days, 5 ml. of thisculture was used to inoculate each 1 liter Erlenmeyer flask containing250 ml. of the various media for test in shake cultures at 24 C. Theyields of inhibitor activity obtained after 96 hours of cultivation wereassayed. Three synthetic media, Czapeks sucrose medium, starch-nitratemedium, lactate medium (Waksman, 1950, pp. 193-197, No. 18, 19, 26) gavelittle inhibitor activity, 0.04 pg. of trypsin inhibited to 50% per l.or less. Growth was also relatively sparse in these media with theexception of the starch-nitrate medium. Higher yields were attained innutrient broth, glucose-peptone medium (Sabouraud), and in a milk medium(Dion, W. M., Can. J. Research, C, 28 (1950) (577585). 0.3 g. of trypsinwas inhibited to 50% per l. The highest yields were attained from amedium containing spray-dried protein powder, which contained (g. perliter in distilled water): protein powder (Bast Bovine Cell Tissue, C.E. Basts Successors Ltd., Copenhagen, Denmark) 18; glucose, 10; K HPO1.0; NaOH 3.0; and MgSO -7H O, 0.5, 1-2 g. of trypsin were inhibited to50% per l.

Example 2 Nutrient agar slants containing the microorganism NRRL 3859were used for direct inoculation of 1 liter Erlenmeyer fiasks containing250 ml. of protein powder medium. The flasks were incubated at 24 C. ona rotary shaker (180 rev./min.). Maximum trypsin inhibition (1 l.inhibited 1.90 g. of trypsin to 50%) and chymotrypsin inhibition (1 l.inhibited 2.29 g. of chymotrypsin to 50%) was attained after 112 hoursof cultivation.

Example 3 Commercial glass-bodied fermentors with stainless-steelequipment and a maximum capacity of 10 liter (Model FL 100, Biotec AB,Bromma, Sweden) were used for cultivation of the microorganism NRRL 3859on an 8 liter scale. In these cases the organism was transferred fromthe nutrient agar slant to a 1 liter Erlenmeyer flask containing 250 ml.of the same medium as was used in the fermentor. After incubation on ashaker as above 100 ml. were used for inoculation of each fermentor. The

Pilot plant equipment, as in Example 3, were used in the cultivation ofthe strain NRRL 3859.

The following conditions were applied to the fermentor cultures: proteinpowder medium; temperature, 28 C.; agitation, 500 rev./min.; aeration,0.6 liter of air per liter of medium per min. Silicone antifoam emulsionRD, diluted 1:10, was added automatically as required. The pH wasprevented from increasing over pH 5 by automatic additions of 0.5 M HCl.Maximum trypsin inhibition (1 l. inhibited 0.90 g. trypsin to 50%) wasobtained after 29 hours of cultivation.

Example 5 Pilot plant equipment, as in Example 3, were used in thecultivation of the strain NRRL 3859.

The following conditions were applied to the fermentor cultures: proteinpowder medium; temperature, 18 C.; agitation, 500 rev./min.; aeration,0.6 liter of air per liter of medium per min. Silicone antifoam emulsionRD, diluted 1:10, was added automatically as required. Maximum trypsininhibition (1 l. inhibited 1.03 pg. trypsin to 50%) was obtained after62 hours of cultivation.

Example 6 Pilot plant equipment, as in Example 3, were used in thecultivation of the strain NRRL 3859.

The following conditions were applied to the fermentor cultures; proteinpowder medium; temperature, 18 C.; agitation 500 rev./min.; areation,0.6 liter of air per liter of medium per min. Silicone antifoam emulsionRD, di luted 1:10, was added automatically as required. The pH wasprevented from increasing over pH 5 by automatic additions of 0.5M HCl,Maximum trypsin inhibition (1 l. inhibited 1.92 g. of trypsin to 50%)was attained after 71 hours of cultivation.

PURIFICATION OF THE PROTEASE INHIBITOR PREPARATION Example 7 Theculture, obtained according to Examples 1-6, was filtered by suction.The culture filtrate was evaporated to a tenth of its volume by vacuum.To the concentrated culture filtrate was added 50% by volume of coldacetone. The precipitate was removed by centrifugation, and the acetonein the supernatant evaporated by vacuum. At least a tenfold purificationof the protease inhibitor fraction contained in the crude culturefiltrate was obtained by this process. The resulting inhibitor solutionwas freezedried with only a moderate loss of activity.

Example 8 The culture, obtained according to Examples l-6, was filteredby suction. After addition of polyvinylpyrolidone (Polyclar AT, 1 g. perml., General Aniline & Film Corp., New York, NY.) the solution wasfreeze-dried. The dry powder was extracted with methanol. After anaddition of one fifth by volume of distilled water to the extract, themethanol was evaporated by vacuum. The remaining solution was pouredinto a dialysis tub, which was placed into distilled water at 5 C.overnight. The

TABLE V [Purification of protease inhibltorggiigim Streptomycesviolascena NRRL Inhibitor activity (percent) Chymo- Trypsiu trypsinTreatment inhibitor inhibitor Filtration of culture 100 100 85 77 69 6663 58 Gel filtration 33 21 PRESERVATION OF HERRING PREPARATION Example9' 110 g. of herring was homogenized in an Ultra Turrax (Janke & KunkelKG) with 110 ml. of distilled water. The suspension was centrifuged for50 min. at 9,000 rpm. The protease activity in the supernatant, with andwithout addition of inhibitors, was assayed as caseolytic activity at pH5.0, 7.2 and 9.5 by a modification of Ansons procedure (Jtinsson, Appl.Microbiol. 15 (1967) 3l9- 324). The inhibitors were added as culturefiltrate with a capacity of inhibiting 1.8 ,ug. of trypsin to 50% per111. Maximal inhibition was attained with an addition of about 250 ml.of the inhibitor solution per kg. of herring, which contained 19.4, 18.6and 23.6 milliunits of protease activity per kg. as estimated at pH 5.0,7.2 and 9.5, respectively. The maximal inhibition was 20, 15 and 70%,respectively, at the various pH values.

INHIBITION OF PROTEOLYTIC ACTIVITY ON A PROTEASE PREPARATION OBTAINED ATCUL- TIVATION OF CEPHALOSPO'RIUM ATCC 11 550.

Example 10 A suitable amount of protease obtained at cultivation ofCephalosporium ATCC 11 550 was dissolved in 0.2 M phosphate buffer of pH7.4. The protease inhibitor preparation according to the presentinvention was dissolved in a separate solution of the same buffer in aconcentration of 10 mg./ml. Thereafter 0.1 ml. of the protease solutionand increasing amounts of the inhibitor solution was mixed and buffer upto 3 ml. was added. The mixture thus obtained was incubated for 15minutes at 37 C., whereafter 3 ml. of a 3% casein solution of pH 7.4 wasadded. Casein analyses forproteolytic activity were carried out asdescribed by Ber'gkvist, Acta Chem. Scand. 17 (1963) 1521-1540. 15 g. ofcasein (Merck, Hammarsten casein) was suspended in 400 ml. of waterunder vigorous stirring. Sodium hydroxide was added until dissolution.The pH was adjusted to the desired pH and the solution was diluted to500 ml. with water. 3 ml. of casein solution was mixed in test tubeswith 3 ml. of the incubated solution mentioned above.

Parallels of the test tubes were used. 2. ml. from the tubes werepipetted immediately into 3 ml. of 10% trichloroacetic acid. The rest ofthe contents of the test tubes were incubated for exactly minuteswhereafter 2 ml. were withdrawn and added to 3 ml. 10% trichloroaceticacid.

After standing for at least 30 minutes at room temperature the tubeswere centrifuged and filtered through small funnels stoppered with alose plug of cotton. The absorbance at 280 nm. of the filteredcentrifugates was measured. Optical pathway: 1 cm.

The difference UV -UV in absorbance, that is the difference inabsorbance measured on samples after 30 minutes incubation, UV andmeasured on samples without incubation, UV is a measure of theproteolytic activity. Thus, decreasing values of UV UV means in--creasing inhibition of proteolytic activity. A solution of 1 mg.protease preparation, obtained from Cephalosporium ATCC 11 550, per ml.was used. 0.1 ml. of this solution was used for the analysis. Theresults are reported in Table VI:

TABLE VI Inhibition of proteolytic activity in protease preparationsobtained from Cephalosporium ATCC 11 550] Absorbance Difference Amountof After 30 At 0 min. inhibitor added min incubaineubaabsorbance tion(Uvso) tion (UVu) UV;o UVo Percent Volume, Weigh t, arbitrary arbitraryarbitrary inhibiml. mg. units units units tion It is seen from theresults given in Table VI that about 0.55 mg. of the tested proteaseinhibitor preparation will cause a 50% inhibition of the proteolyticactivity in 0.1 mg. of the Cephalosporium protease preparation.

INHIBITION OF PROTEOLYTIC ACTIVITY ON A BERGKVIST PROTEASE I PREPARATIONOB- TAINED BY CULTIVATION OF ASPERGILLUS ORYZAE Example 11 TABLE VII[Inhibition of proteolytic activity in protease preparations of ProteaseI obtained from Aspergillus Oryzae] Absorbance Difference Amount ofAfter 30 At 0 min. in

inhibitor added min. ineuba ineubaabsorbance tion (UVao) tion (UVo)UVso- UVo Percent Volume, Weight, arbitrary arbitrary arbitraryinhibiml. mg. units units units tion It is seen from the resultsreported in Table VII that about 0.55 mg. of the protease inhibitorpreparation will cause 50% inhibition of the proteolytic activity in0.030 mg. of the Protease I preparation.

Thus, according to the present invention, a commercially acceptablemethod for producing protease inhibitors in large quantities isprovided. Many variations of this in- 'vention will readily suggestthemselves to those skilled in the art. In this regard nothing in thepreceding disclosure is intended to limit the scope of the invention asdefined by the following claims.

We claim:

1. A process for preparing a preparation containing protease inhibitorswhich comprises cultivating Streptomyces violascens, NRRL 3859, underaerobic conditions in a nutrient medium containing sources of carbon,nitrogen, and nutrient inorganic salts in assimilable form, for a periodof about 10 to 100 hours until a sutficient amount of proteaseinhibitors is produced, said medium having a pH, prior to inoculation,ranging between about 5 and 8, and a temperature during fermentationranging from about 10 to about 40 C.

2. A protease inhibitor preparation prepared by the process according toclaim 1.

12 References Cited Miller: The Pfizer Handbook of MicrobialMetabolites, McGraw-Hill Book Co., Inc., New York, N.Y., 1961, pp. 394,611 and 612.

JEROME V. GOLDBERG, Primary Examiner US. Cl. X.R. 19580

1. A PROCESS FOR PREPARING A PREPARATION CONTAINING PROTEASE INHIBITORSWHICH COMPRISES CULTIVATING STREPTOMYCES VIOLASCENS, NRRL 3859, UNDERAEROBIC CONDITIONS IN A NUTRIENT MEDIUM CONTAINING SOURCES OF CARBON,NITROGEN, AND NUTRIENT INORGANC SALTS IN ASSIMILABLE FORM, FOR A PERIODOF ABOUT 10 TO 100 HOURS UNTIL A SUFFICIENT AMOUNT OF PROTEASEINHIBITORS IS PRODUCED, SAID MEDIUM HAVING A PH, PRIOR TO INOCULATION,RANGING BETWEEN ABOUT 5 AND 8, AND A TEMPERATURE DURING TERMENTATIONRANGING FROM ABOUT 10* TO ABOUT 40*C.